Team:UT-Dallas/Project/methods

Methods and Protocols

Project

Miniprep

We used QIAGEN's miniprep reagents and protocol.

1. Pellet cells at 4000 rpm, 4C for 12 minutes
2. Resuspend cells with 200ul buffer P1, transfer to microcentrifuge
3. Add 200 ul lysis buffer P2, cap and shake. Do not allow reaction to continue for more than 5 minutes.
4. Add 300 ul Neutralization buffer N3, cap and shake.
5. Pellet cells at 13000 rpm for 10 minutes with tabletop centrifuge.
6. Apply supernatant to spin column.
7. Centrifuge for 60s. Discard flow through.
8. Add 750 ul wash buffer PE to column.
9. Centrifuge for one minute. Discard flow through.
10. Centrifuge again for one minute to get rid of excess wash buffer.
11. Transfer column to microcentrifuge tube. Add 50 ul elution solution EB to the column, let stand for 1-5 minutes.
12. Centrifuge for 60s. Determine concentration of DNA product and store at -20C

Gel Extraction

We used QIAGEN's Gell extraction protocol and reagents.

1. Use razor to excise gel slice containing desired DNA.
2. Slice this gel finely and place in a microcentrifuge tube.
3. Add 750 ul QG buffer to tube.
4. Incubate at 50C while shaking tubes at 500 rpm. You may need to vortex the tubes occasionally. Continue until the gell has completely dissolved. This is usually around 10 min.
5. Add something

Transformation Protocol

1. Thaw competent cells on ice.
2. To a microcentrifuge tube, add 50ul cells if using homestock, 25ul if using commercial cells.
3. Add 1-2 ul of desired DNA.
4. Incubate on ice for 30 min.
5. Heat shock at 42C for 30 sec.
6. Ice shock cells for 2 min.
7. Add 450 ul SOC broth media.
8. Incubate at 30C while shaking at 300 rpm for 60 min.
9. Incubate appropriate antibiotic plates at 37C.
10. Plate cells. Incubate overnight at 37C.

Inoculation for Miniprep

1. Grow cells overnight on plates at 37C
2. Circle desired colonies with pen.
3. Use a pipette tip to scrape colonies off the plate.
4. Place pipette tip in falcon tube with 3ml of appropriate antibiotic broth.
5. Incubate at 37C, while shaking at 1000 rpm overnight.

Dinosaur

This is a dinosaur!!

Some Other Protocol

Well, aren't we interesting

More about our project:

• History of Cholera
• Project Details
• Materials and Methods
• The Experiments
• Results
• Data analysis
• Conclusions